Enzymatic process



latented June 14, 1960 its ENZYMATL'C FROCESS Donald W. Ohlmeyer, Glenview, 111., assignor to Ben L. Sarett, Chicago, Ili.

N Drawing. Filed Apr. 24, 1957, Ser. No. 654,677

8 Claims. (Cl. 195-66) This invention relates to an enzymatic process and has for an object the provision of a process for removing proteolytic enzymes from a glucose oxidase-containing enzyme system.

Glucose oxidase is a well known enzyme which may be prepared by known methods from the mycelia of various microorganisms, such as molds of the genus Aspergillus and Penicilium. One procedure for producing glucose oxidase is disclosed in Dwight L. Baker Reissue Patent No. 23,523, dated July 22, 1952. As pointed out in the Baker reissue patent and as pointed out in Robert R. Baldwin Patent No. 2,744,017, dated May 1, 1956, the enzyme glucose oxidase has found many practical applications particularly in connection with the deoxygenation or desugarizing of certain foods and food products. In accordance with certain of the procedures utilizing the glucose oxidase enzyme system, the glucose oxidase (usually containing catalase) is dispersed directly within the product to be treated, thereby providing means for the direct removal of oxygen or sugar as the case may be from the medium within which it is dispersed.

Commercial preparations of glucose oxidase usually contain the enzyme catalase and have also been found in many instances to contain substantial amounts of proteolytic enzymes. In many types of food treatment such proteolytic enzymes are not objectionable, particularly when the product contains no substantial quantities of proteinaceous material or where the product is dried shortly after treatment. However, there 'are certain protein-containing food products which are susceptible to treatment with glucose oxidase for known purposes but which may be adversely afiected by the proteolytic enzymes which may be found as impurities in commercial glucose oxidaseenzym'e preparations under conditions wherein the food products contain water ,and are stored in the presence of water for long periods of time in which For exthe proteolytic enzymes are given time to act. ample, it has been found in certain instances that if glucose oxidase is used in the treatment of milk products any proteolytic enzyme that may be present as an impurity in the glucose oxidase may serve upon storage in the presence of water to convert the casein to paracasein whereby the aqueous milk product will be coagulated or have an undesired tendency to coagulate. Furthermore, if the enzyme system is used in connection with the treatment of products containing substantial amounts of proteinaceous. materials, any proteolytic enzyme that may be introduced by way of the glucose oxidase enzyme prepara tion may have a tendency upon storage of the product in the presence of water to degrade the structure of the protein. Thus gelatin products will be adversely affected in that the gelatin will not gel properly and the product thus treated is unsuitable for ready customer acceptance.

Accordingly, a further object of this invention is the provision of a method for separating or removing proteolytic enzymes that may be present in commercial glucose oxidase preparations.

A still further object of thisinvention is the provisionof a method of removing proteolytic enzymes from glucose oxidase preparations without substantially reducing the potency of the glucose oxidase contained therein and without materially aiiecting the potency of the catalase that may also be present in such preparations. 'i

A still further object of this invention is the provision of a process for readily preparing a glucose oxidase composition which may contain catalase but which is essentially free from proteolytic enzymes.

'Further and additional objects will appear from-the following description and accompanying claims.

In accordance with one embodiment of this invention, it has been found that proteolytic enzymes may be removed from an enzyme preparation containing glucose oxidase by heating an aqueous solution or dispersion of the enzyme preparation under controlled conditions whereby the proteolytic enzyme is preferentially deac-v tivated without substantially affecting the activity of the glucose oxidase or of any catalase that may be associated therewith. The invention is primarly applicable to concentrated aqueous dispersions of the glucose oxidasc-containing enzyme system and is more particularly applicable to dispersions which have a concentration of glucose oxidase in excess of about 200 units per gram. A unit of glucose oxidase is herein intended to mean the unit essentially as defined in Dwight L. Baker. Patent No. 2,651,592, dated September 8, 1953. The concentrated glucose oxidase dispersions may include any of those that are presently commercially available. Certain of these are available in the aqueous solution state. Others are in the form of dry powders which may be dispersed in water prior to treatment in accordance with this invention. The temperatures useful for preferentially removing the proteolytic enzymes is between about 50 and C. Below 50 C. the proteolytic enzyme is not inactivated in any reasonable period of time and above about-80 C. (particularly above about 87 C.) the glucose oxidase itself is rapidly destroyed. In commercial operations it is preferable to operate at temperatures between about 55 and 65 C. The time during which the heating is carried out may vary widely depending upon the concentration of the enzyme, the temperatures employed and the hydrogen-ion concentration. Broadly speaking, the time of treatment may vary between about 5 minutes and 10 hours, a shorter period of time being applicable when using the higher temperatures. For

most practical operations, however, the time of heating is usually between about 15 minutes and 2 hours- 'With respect to the hydrogen-ion concentration, it is usually preferred to operate between pH 5 and 7. The pH range which represents the broadest range in which the process may be carried out is usually in the area of pH 4 to 8.

After the aqueous dispersion of the enzyme system has been heated under the conditions above indicated, the proteolytic enzyme will be inactivated and will usually form as a precipitate. This precipitate may be removed by settling, filtering or centrifuging and the resulting glucose oxidase solution, the normal catalase content thereof being substantially unimpaired, can be used in the usual manner to treat food or otherproducts as is well known.

For a more complete understanding of this invention, reference will now be made to the following examples showing specific methods for removing proteolytic enzymes from glucose oxidase preparations.

Example 1 A commercially available glucose oxidase preparation containing catalase and proteolytic enzymes in the form of a clear amber aqueous solution containing about 750 units of glucose oxidase per milliliter was heated to 1 "A expel-anthems eanieeipui il'tiiizmg 1 ie eg;

avast 60" c. for so minstes.

tion was essentially free of proteolytic enzyme still had a glucoseroxidase activity of 725 units per m lliliter.

contained mass; in 'sub'stant iaily undiminished l't'i's suitable for us'e'in the treatment of various types of protein-containing products for des'ugarizin'g for d e- ,n ox genmn as is wen them, 7 I

7 V ExampleiZh I V I mere-e119 available gin'cose axiaas'ssrepmt-gon infthe fo rm of a solution of dispe cataiaserand' ii pfcfeetygieisz nie was hes-{ea at 60 for time hour; The wereiinen tio'n'an the resultin s and unless friimw ftoivti'enzymt-fhada remand?" 111- this instance-the activity arne ginaiiam was reamed am abate '-8 pereeatan ebit-he heatin -amongst; ofthenroteoiyti its-em nent resist/ea? eaesz; e useful for treating 'Eo'od'br dihifl matinee men would In order re aembnsifate the eaeetivehss bfftbe erein disclosed procedure inr'einoving'proteolytic enzyme from commercial-'giuco'se oxidase preiaaraft ions, a gelatin soluftion was prepare by dissolvingi grams of gelatin in "500 milliliters ofjwarm'wa'te'r. The'soiut'ionwas divided txi'n'to three boi'tions of 130 milliliters eaeh While still warm 2O milliliters of glucose oxidase treated in ac- 'cordance with Examplel were added to "a first pm-trail, 26 milliliters "of the glucose oiiidase indicated in Exam le fl but 'which was not treated were added to a second ier} ftiom and ZOQfiiiHiIiters of water were'addfe'd to "af'thiid gsrtipm ime resulting three'inixt'ui'es were allowed to s and at roam tempe amres rm one hour h athen :13 at in a i'efrigefator at 40 F.- After six 'day'sitfwas no' teii firm er a rigid geI'vJhiIejhesecbnH port on did nbt'gl and was soft anziwaie'ry. is proof that me same erson of gmcdse ldxida'se tesmsineas prsee- "tyne r-sniymewhich v'gas Tammany the treattiin been ais'claseat-r K lavense we tsth m I wing; a coagiflatioh milk" prot ,7 fiparatien had b Phe glucose axiaase product'tre'ated'in a "the process oi this inventidnflreinain's substantiallyuncosethfei'efrom in accordance with the 'proceduresj'se't forth' in Baker 'Reisfs u'elljatent -No.[2'3,s23 and Baldwin.

The 'prb'duct, being free of smteclync enzyme, net

) y ei ir'fstance the "t n- 7 l-exhibited a tendency mesa-gum the] r hile the "treated; glucose snag-s ecbtaanekvim respect to its glucose oxidaseand icatalase content and it is particularly useful in the treatment of V A precipitate formed which wasseparated 'by filtration. The resulting soluadversely affect a protein-containing substance treated with it even though that substance-be stored over long periods of time in the presence of water. Thus the protein does not degrade or otherwise change due to proteolytic enzyme activity. a 7

While a particular embodiment of this invention is shown above, it will be understood of course, that the invention is not to be limited thereto, since many modi- 7 fications may be made, and it' is contemplated, therefore,

'11 containing:- ab out 7 sooo'nnitsir iuwse eiia'ase ser milliliter as iveiiasby the appended claims, to cover any such modifications as fall the true spirit and scape of this invention. a I claim: 7 I

I. A- process for removing proteolytic enzymes from a glucose oxidase enzyme system containing the same which comprises the steps of heating an aqueous dispersion of said enzyme system having a pH between about 4 and about 8 to a temperature between about 50 and 80 C. for a period of time tsufiicientto inactivate a sub- "stiint'ifil portion of'said prdt'eolyti 1 Thepr es a system n'neaea ref eea'abqsc east-smegma 7 10 hours.

3. A process at removing 'firbteolyt" sieves 1-is; The processrecitedin nan- 4 in when sintins between about 1-5 minutes ans 2 hears; 1

'6. "me recited in exam t in 'e iifi is between about 5 and 7.

, 7; "A presets for a glucose oiiiiiis iiiyfitiining enzyme s stem which "compn es steps f aqueous selutie'n of "Said enz me-"s stem ;Patent,No. 2,744,0 1 7. The amounts of'thergiucose 'oXiease product employed in each instance will depend the iisual time, tempe rature and} bbilceiitl' a tidn fgctbrgfiswell known and as diseases in said patents.

:peramre between-about and so" is: a

and having a pH between about d and seem a is a of nine sumeiem to tame-"preci itate but insane eh't in destro" y a substam tial emnant of megmeeseanaase ires- 7 s; The process new! "I'Wh'ti'n saiti cipi't ate is sees-raise the aestea sewa e.

References frm-r'TEn sTATEsPXTErt'rST 2,883,681 MiHe: tai. '.;;.,-;e. iuly 1-3, oman REFERENCES Che-matey and *racn miagy of :raaiaei,

."1949, ub. b'y mm Wiley 'anasbhs, me. (New York), b t- Chemistry ands/tastiest e'hyif f ra-i 1953. pub. by Aeaaeme mess ins. Y'erk pige'tt. 

1. A PROCESS FOR REMOVING PROTEOLYTIC ENZYMES FROM A GLUCOSE OXIDASE ENZYME SYSTEM CONTAINING THE SAME WHICH COMPRISES THE STEPS OF HEATING AN AQUEOUS DISPERSION OF SAID ENZYME SYSTEM HAVING A PH BETWEEN ABOUT 4 AND ABOUT 8 TO A TEMPERATURE BETWEEN ABOUT 50* AND 80*C. FOR A PERIOD OF TIME SUFFICIENT TO INACTIVATE A SUBSTANTIAL PORTION OF SAID PROTEOLYTIC ENZYME. 